Toxicology and efficacy of tumor-targeting Salmonella typhimurium A1-R compared to VNP 20009 in a syngeneic mouse tumor model in immunocompetent mice.

Salmonella typhimurium A1-R (S. typhimurium A1-R) attenuated by leu and arg auxotrophy has been shown to target multiple types of cancer in mouse models. In the present study, toxicologic and biodistribution studies of tumor-targeting S. typhimurium A1-R and S. typhimurium VNP20009 (VNP 20009) were performed in a syngeneic tumor model growing in immunocompetent BALB/c mice. Single or multiple doses of S. typhimurium A1-R of 2.5 × 105 and 5 × 105 were tolerated. A single dose of 1 × 106 resulted in mouse death. S. typhimurium A1-R (5 × 105 CFU) was eliminated from the circulation, liver and spleen approximately 3-5 days after bacterial administration via the tail vein, but remained in the tumor in high amounts. S. typhimurium A1-R was cleared from other organs much more rapidly. S. typhimurium A1-R and VNP 20009 toxicity to the spleen and liver was minimal. S. typhimurium A1-R showed higher selective targeting to the necrotic areas of the tumors than VNP20009. S. typhimurium A1-R inhibited the growth of CT26 colon carcinoma to a greater extent at the same dose of VNP20009. In conclusion, we have determined a safe dose and schedule of S. typhimurium A1-R administration in BALB/c mice, which is also efficacious against tumor growth. The results of the present report indicate similar toxicity of S. typhimurium A1-R and VNP20009, but greater antitumor efficacy of S. typhimurium A1-R in an immunocompetent animal. Since VNP2009 has already proven safe in a Phase I clinical trial, the present results indicate the high clinical potential of S. typhimurium A1-R

Extensive Hair Shaft Growth after Mouse Whisker Follicle Isolation, Cryopreservation and Transplantation in Nude Mice.

We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles

Extensive Hair-Shaft Elongation by Isolated Mouse Whisker Follicles in Very Long-Term Gelfoam® Histoculture

<div><p>We have previously studied mouse whisker follicles in Gelfoam® histoculture to determine the role of nestin-expressing plutipotent stem cells, located within the follicle, in the growth of the follicular sensory nerve. Long-term Gelfoam® whisker histoculture enabled hair follicle nestin-expressing stem cells to promote the extensive elongation of the whisker sensory nerve, which contained axon fibers. Transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP) were used as the source of the whiskers allowing imaging of the nestin-expressing stem cells as they formed the follicular sensory nerve. In the present report, we show that Gelfoam®-histocultured whisker follicles produced growing pigmented and unpigmented hair shafts. Hair-shaft length increased rapidly by day-4 and continued growing until at least day-12 after which the hair-shaft length was constant. By day-63 in histoculture, the number of ND-GFP hair follicle stem cells increased significantly and the follicles were intact. The present study shows that Gelfoam® histoculture can support extensive hair-shaft growth as well as hair follicle sensory-nerve growth from isolated hair follicles which were maintained over very long periods of time. Gelfoam® histoculture of hair follicles can provide a very long-term period for evaluating novel agents to promote hair growth.</p></div

Long-Term Extensive Ectopic Hair Growth on the Spinal Cord of Mice from Transplanted Whisker Follicles

<div><p>We have previously demonstrated that hair follicles contain nestin-expressing pluripotent stem cells that can effect nerve and spinal cord repair upon transplantation. In the present study, isolated whisker follicles from nestin-driven green fluorescent protein (ND-GFP) mice were histocultured on Gelfoam for 3 weeks for the purpose of transplantation to the spinal cord to heal an induced injury. The hair shaft was cut off from Gelfoam-histocultured whisker follicles, and the remaining part of the whisker follicles containing GFP-nestin expressing pluripotent stem cells were transplanted into the injured spinal cord of nude mice, along with the Gelfoam. After 90 days, the mice were sacrificed and the spinal cord lesion was observed to have healed. ND-GFP expression was intense at the healed area of the spinal cord, as observed by fluorescence microscopy, demonstrating that the hair follicle stem cells were involved in healing the spinal cord. Unexpectedly, the transplanted whisker follicles sprouted out remarkably long hair shafts in the spinal cord during the 90 days after transplantation of Gelfoam whisker histocultures to the injured spine. The pigmented hair fibers, grown from the transplanted whisker histocultures, curved and enclosed the spinal cord. The unanticipated results demonstrate the great potential of hair growth after transplantation of Gelfoam hair follicle histocultures, even at an ectopic site.</p></div

Hair shaft elongation of mouse whiskers in Gelfoam^® histoculture.

<p>Time-course images of hair shaft growth from individual mouse whisker follicles, isolated from nestin-driven green fluorescent protein (ND-GFP) mice, histocultured on Gelfoam®. Green fluorescence was from the ND-GFP-expressing stem cells in the whisker hair follicles which were enriched during 63 days of histoculture <i>in vitro</i>. Hair shafts lengthened rapidly in the first 4 days, extended over 9–12 days, and remained the same length until day 63.</p

Graph quantifying the time-course increase of hair follicle stem cell GFP fluorescence intensity (A) and fluorescent area (B).

<p><i>p</i><0.01 in increase of fluorescent area and fluorescence intensity at day 63 compared to day 1.</p

Graphs quantifying the increase of shaft length over time in individual follicles during Gelfoam® histoculture.

<p>Graphs quantifying the increase of shaft length over time in individual follicles during Gelfoam® histoculture.</p

Ectopic hair growth in the spinal cord.

<p>Ninety days after transplantation of the 3-week Gelfoam ND-GFP-expressing whisker histocultures in the injured spinal cord, long hair shafts (arrows), were observed along and around the healed spinal cord. (<b>A</b>) Shows the elongated hair shafts that grew from whisker follicles, previously histocultured on Gelfoam into the injured spinal cord in 3 different mice at day-90 after surgery. All mice demonstrated hair shaft growth from the transplanted histoculture whisker follicles. Mouse 3 had the most remarkable hair shaft growth, which curved and enclosed the spinal cord. Arrows showed the hair growth in the spinal cord. (<b>B</b>) Panels show the hair shaft growth from the transplanted Gelfoam histoculture whisker follicles in the spine from mouse 3 at higher magnification from different views of the spinal cord (dorsal, left, and right side). The growing hair shaft reached a length of almost 14 mm and curved around the spinal cord. Arrows depict the hair shaft growing from the whisker hair follicles transplanted in the spine. Six out of 7 mice implanted with the Gelfoam whisker histoculture showed extensive ectopic hair growth on the spine.</p